LOCUS       sequence-390570-        5461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert to create split-intein Gal4[N-int] knock-in vectors via 
            Drop-In cloning, released via BsmBI digest .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5461)
  AUTHORS   Ewen-Campen B, Luan H, Xu J, Singh R, Joshi N, Thakkar T, Berger B, 
            White BH, Perrimon N
  TITLE     split-intein Gal4 provides intersectional genetic labeling that is 
            repressible by Gal80.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Jun 13;120(24):e2304730120. doi: 
            10.1073/pnas.2304730120. Epub 2023 Jun 5.
  PUBMED    37276389
REFERENCE   2  (bases 1 to 5461)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2023-06-13"; volume: "120(24):e2304730120. 
            doi"; pages: " 10.1073/pnas.2304730120. Epub 2023 Jun 5"
COMMENT     Sequence Label: pDropIn-T2A-split-intein-Gal4[1-20]-N-int
FEATURES             Location/Qualifiers
     source          1..5461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             103..405
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     284..303
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             754..1548
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             754..1548
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(808..827)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1418..1437
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     regulatory      1748..1757
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1754..2128
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      2266..2854
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2755..2774
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3008..3025
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3142..3163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3178..3183,3184..3201,3202..3208)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3216..3232
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3221..3243
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3240..3256
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3240..3256
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3274..3292
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3274..3291
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             3410..3463
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     protein_bind    4048..4081
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      4307..4316
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4313..4990
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(4443..4461)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4493..4513)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4711..4730
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4905..4928
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    5111..5232
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5148..5167)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5202..5221
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    5262..5295
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5384..5403)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5385..5403)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5410..5427)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5410..5426)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5419..5441)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
       61 accgatcgcc cttcccaaca gttgcgcagc ctatacgtac ggcagtttaa ggtttacacc
      121 tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg
      181 ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc
      241 cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat
      301 atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa
      361 aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc aggcatgaga
      421 ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc gcagaaacgg
      481 tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa cgcaagcgca
      541 aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg ggcggtttta
      601 tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
      661 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaagc
      721 tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
      781 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
      841 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
      901 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
      961 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     1021 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     1081 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     1141 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     1201 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     1261 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     1321 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     1381 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     1441 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     1501 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat tattaacgct
     1561 tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcatc
     1621 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1681 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatagcacgt
     1741 gaggagggcc accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt
     1801 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga
     1861 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca
     1921 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc
     1981 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga
     2041 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt
     2101 gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa cttcattttt aatttaaaag
     2161 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2221 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2281 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2341 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     2401 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     2461 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     2521 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     2581 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     2641 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     2701 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     2761 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     2821 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     2881 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     2941 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3001 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3061 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3121 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     3181 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     3241 aggaaacagc tatgaccatg attacgccaa gctatttagg tgacactata gaatactcaa
     3301 gctatgcatc aagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg
     3361 aattcgccct taaactgaga cgggtggaag cggaggtagc gcggccgccg agggccgcgg
     3421 cagcctgctg acctgcggcg atgtggagga gaaccccggg cccgctagca tgaagctact
     3481 gtcttctatc gaacaagcat gcgatatttg ccgacttaaa aagctcaagt gcctggatct
     3541 gaagacccag gtgcagaccc cccagggcat gaaggagatc agcaacatcc aggtgggcga
     3601 tctggtgctg agcaacaccg gctacaacga ggtgctgaac gtgttcccca agagcaagaa
     3661 gaagagctac aagatcaccc tggaggatgg caaggagatc atctgcagcg aggagcacct
     3721 gttccccacc cagaccggcg agatgaacat cagcggcggc ctgaaggagg gcatgtgcct
     3781 gtacgtgaag gagtaacctc tagagtcgac taaagccaaa tagaaattat tcagttctgg
     3841 cttaagtttt taaaagtgat attatttatt tggttgtaac caaccaaaag aatgtaaata
     3901 actaatacat aattatgtta gttttaagtt agcaacaaat tgattttagc tatattagct
     3961 acttggttaa taaatagaat atatttattt aaagataatt cgtttttatt gtcagggagt
     4021 gagtttgctt aaaaactcgt ttggatcata acttcgtata atgtatgcta tacgaagtta
     4081 tcgtacggga tctaattcaa ttagagacta attcaattag agctaattca attaggatcc
     4141 aagcttatcg atttcgaacc ctcgaccgcc ggagtataaa tagaggcgct tcgtctacgg
     4201 agcgacaatt caattcaaac aagcaaagtg aacacgtcgc taagcgaaag ctaagcaaat
     4261 aaacaagcgc agctgaacaa gctaaacaat cggctcgaag ccggtcgcca ccatggcctc
     4321 ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa
     4381 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
     4441 cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca tcctgtcccc
     4501 ccagttccag tacggctcca aggtgtacgt gaagcacccc gccgacatcc ccgactacaa
     4561 gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     4621 cgtggtgacc gtgacccagg actcctccct ccaggacggc tccttcatct acaaggtgaa
     4681 gttcatcggc gtgaacttcc cctccgacgg ccccgtaatg cagaagaaga ctatgggctg
     4741 ggaggcgtcc accgagcgcc tgtacccccg cgacggcgtg ctgaagggcg agatccacaa
     4801 ggccctgaag ctgaaggacg gcggccacta cctggtggag ttcaagtcca tctacatggc
     4861 caagaagccc gtgcagctgc ccggctacta ctacgtggac tccaagctgg acatcacctc
     4921 ccacaacgag gactacacca tcgtggagca gtacgagcgc gccgagggcc gccaccacct
     4981 gttcctgtag gggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt
     5041 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     5101 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5161 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5221 caatgtatct taaccgtctg taagcagcga aggcccatac gataacttcg tataatgtat
     5281 gctatacgaa gttatgagct ccacgtctct cgcgaagggc gaattctgca gatatccatc
     5341 acactggcgg ccgctcgagc atgcatctag agggcccaat tcgccctata gtgagtcgta
     5401 ttacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5461 a
//
